Scanning Tunneling Microscopy of DNA
DNA is looked upon as the most critical target of ionizing radiation in living cells. The prominent types of lesions are DNA single (SSBs) and double strand breaks (DSBs). The DNA double-strand break (DSB) is regarded as the essential type of lesion. Both SSBs and DSBs are introduced and resealed regularly by enzymatic processes during the life cycle of cellular DNA. Incorrect repair of radiation induced DSBs may result in hazardous consequences like mutations, transformations or even mitotic death of the cell. However, DSBs do not represent an uniform species of damage. Either the phosphate or the sugar moiety may be damaged, and the spatial distribution of the two opposite ruptures in the sugar-phosphate backbone varies, too.
In the last years, radiation research has focused on the unveiling of the exact molecular nature of radiation induced DNA damage. Many different experimental methods are in use today to detect and quantify DNA double-strand breaks, but all these methods have in common the inability to differentiate specific types of lesions in individual molecules. They only detect effects by integration over many identical molecules (> 10^7), but qualitative differences in the damage induction as proposed between low- and high-LET radiation can not be detected with conventional experimental methods currently available. Refined microscopic techniques like Scanning Tunneling Microscopy (STM) offer new possibilities.
Applying Scanning Tunneling Microscopy, the possibility of imaging individual molecules and getting a realistic visual impression of radiation defects was investigated. A pocked-sized STM was employed which provides atomic resolution. DNA was dissolved in aqueous solution, dropped onto a layer of highly oriented graphite and dried in air. Tunneling tips consisted of tungsten-wires that were mechanically cut or chemically etched. Scanning Tunneling Microscopy of the specimen was always performed under atmospheric conditions to avoid influences onto the biological sample.
(You may download it, provided you refer to the author and GSI when published. Unfortunately we don't have this picture with a better resolution, this was a limitation of the data taking procedure)
With purified salmon sperm DNA, high resolution images were obtained allowing the determination of the dimensions of the DNA double helix. The apparent diameter of the DNA molecule shown here is about 2 nm, in excellent agreement with the expected value. The molecule is right-handed and the helix pitch amounts to 3.3 nm.
The primary purpose of our investigations is the visualisation of DNA lesions. For further experiments, focusing on the observation of X-ray induced damages, plasmid DNA was used as specimen. After X-irradiation significantly conformational changes could be observed with STM. With technical advances, STM may become a versatile analytical requisite for the qualitative analysis of high- and low-LET DNA damage on the molecular level.
Contact: Wolfgang Schonert, original author. Since he has left our group, in case of emergency: M.Kraemer@gsi.de